Method of differentiating microbial colonies in an image

ABSTRACT

A method of identifying microbial colonies in a culture device is provided. The method comprises using an imaging device to produce a first image of a thin film culture device while providing illumination to a front side of the device and to produce a second image of the thin film culture device while providing illumination to a back side of the device. The method further comprises analyzing the first and second images to identify microorganism colonies in each image, analyzing the first and second images values of a size parameter for a colony at a particular location in the culture device, and comparing the values. The method can be used to differentiate and count at least two colony types.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofPCT/US2013/074882, filed Dec. 13, 2013, which claims priority to U.S.Provisional Patent Application No. 61/739,786, filed Dec. 20, 2012, thedisclosure of which is incorporated by reference in its entirety herein.

BACKGROUND

Biological safety is a paramount concern in modern society. Testing forbiological contamination in foods or other materials has become animportant and often mandatory requirement for developers anddistributors of food products. Biological testing is also used toidentify bacteria or other agents in laboratory samples such as bloodsamples taken from medical patients, laboratory samples developed forexperimental purposes, and other types of biological samples. Varioustechniques and devices can be utilized to improve biological testing andto streamline and standardize the biological testing process.

A wide variety of culture devices have been developed. As one example,culture devices have been developed by 3M Company (hereafter “3M”) ofSt. Paul, Minn. In particular, culture devices are sold by 3M under thetrade name PETRIFILM plates. Culture devices can be utilized tofacilitate the rapid growth and detection of microorganisms commonlyassociated with food contamination, including, for example, aerobicbacteria, E. coli, coliform, enterobacteria, yeast, mold, Staphylococcusaureus, Listeria, Campylobacter, and the like. The use of PETRIFILMplates, or other growth media, can simplify bacterial testing of foodsamples.

Culture devices can be used to enumerate or identify the presence ofbacteria so that corrective measures can be performed (in the case offood testing) or proper diagnosis can be made (in the case of medicaluse). In other applications, culture devices may be used to rapidly growmicroorganisms in laboratory samples, e.g., for experimental purposes.

Biological scanning units refer to devices used to scan and/or countmicrobial colonies. For example, a food sample or laboratory sample canbe placed on a culture device, and then the plate can be inserted intoan incubation chamber. After incubation, the culture device can beplaced into the biological scanning unit for automated detection andenumeration of bacterial growth. In this manner, biological scanningunits automate the detection and enumeration of microbial colonies in aculture device, and thereby improve the biological testing process byreducing human error.

SUMMARY

In general, the present disclosure is directed to a technique fordistinguishing objects in a scanned image. In particular, the techniqueis used to differentiate two microorganism colony types that are presentin a culture medium that includes two indicator compounds with whicheach type of microorganism may react. In addition, the technique furthermay be used to count the number of colonies of each microorganism typein the scanned image of the culture device. To count the colonies, aculture device containing the culture medium is inserted into a scanningunit. Upon insertion of the culture device, the scanning unit generatesan image of the culture device. Then, the number of microorganismcolonies can be counted or otherwise determined using image processingand analysis routines performed either within the scanning unit or by anexternal computing device, such as a desktop computer, workstation orthe like. In accordance with the invention, a method of distinguishingcolony types is described. The method can be used to improve theaccuracy over existing methods of automated counts of microorganismcolonies in a scanned image.

In one aspect, the present disclosure provides a method. The method cancomprise using an imaging device to produce a first image of a thin filmculture device, the culture device having a front side having atransparent film cover sheet and a back side having a translucentsubstrate; using the imaging device to produce a second image of thethin film culture device, wherein the second image is produced whileproviding illumination to the back side of the device; analyzing thefirst and second images to identify microorganism colonies in eachimage; analyzing the first image to calculate a first value of a sizeparameter for a colony at a particular location in the culture device;analyzing the second image to calculate a second value of the sizeparameter at the particular location in the culture device; andcomparing the first value to the second value. The first image isproduced while providing illumination to the front side of the device.The culture device comprises first and second indicator compounds,wherein the first indicator compound is converted by a microorganism toa first product having a first color, wherein the second indicatorcompound is converted by a microorganism to a water-diffusible secondproduct that forms a second color.

In any of the above embodiments, the first image can be produced whileilluminating the device with a first ratio of front-side illumination toback-side illumination and the second image can be produced whileilluminating the device with a second ratio of front-side illuminationto back-side illumination that is lower than the first ratio. In someembodiments, the first ratio can be greater than 1:1. In someembodiments, the first ratio can be about 100%:0%. In some embodiments,the second ratio can be about 0%:100%.

In any of the above embodiments, the method further can comprise usingthe first or second image to count a number of microorganism colonies inthe culture device. In any of the above embodiments, the method furthercan comprise using the first and second image to count a number of firsttype colonies and count a number of second type colonies in the culturedevice. In some embodiments, the first type colonies can convert thefirst indicator to the first product. In some embodiments, the secondtype colonies convert the second indicator to the second product.

In any of the above embodiments, the first indicator compound cancomprise a tetrazolium dye. In any of the above embodiments, the secondindicator compound can comprise a chromogenic enzyme substrate thatincludes an indolyl group. In any of the above embodiments, the sizeparameter can be an observed colony diameter. In some embodiments, thecolony diameter can be a colony minimum diameter.

In another aspect, the present disclosure provides a computer readablemedium comprising computer readable instructions that, when executed bya processor, can cause a culture plate scanning system comprising theprocessor to obtain a first image of a thin film culture device, whereinthe first image is produced while illuminating the device with a firstratio of front-side illumination to back-side illumination; obtain asecond image of the thin film culture device, wherein the second imageis produced while illuminating the device with a second ratio offront-side illumination to back-side illumination that is lower than thefirst ratio; analyze the first and second images to identifymicroorganism colonies in each image; analyze the first image tocalculate a first value of a size parameter for a colony at a particularlocation in the culture device; analyze the second image to calculate asecond value of the size parameter at the particular location in theculture device; and compare the first value to the second value. In anyembodiment, the computer readable medium further can comprise computerreadable instructions that, when executed in the processor cause thesystem to use the first or second image to count a number ofmicroorganism colonies in the culture device. In any embodiment, thecomputer readable medium further can comprise computer readableinstructions that cause the system to use the first and second image tocount a number of first type colonies and count a number of second typecolonies in the culture device.

Various aspects of the invention may provide a number of advantages. Forexample, the methods of the present disclosure may improve the accuracyof automated counts of microbial colonies on a culture device. Inparticular, the rules described herein may address problems thatcommonly occur, and which can otherwise undermine the accuracy ofautomated counting of agents on a growth plate.

Additional details of these and other embodiments are set forth in theaccompanying drawings and the description below. Other features, objectsand advantages will become apparent from the description and drawings,and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a perspective view of an exemplary system comprising ascanning device coupled to an external computer which performs imaginganalysis of the images generated by the scanning device.

FIG. 2 is a block diagram of a biological scanning system that maycorrespond to the system illustrated in FIG. 1.

FIG. 3 is a flow diagram illustrating a process of automated analysis ofa microbial culture device.

FIG. 4 is a block diagram of one embodiment of a method of analyzing amicrobial culture device according to the present disclosure.

FIG. 5 is a flow diagram of a counting rule to distinguish microbialcolony types according to the present disclosure.

FIG. 6 is a black and white image of a portion of a thin film culturedevice having two types of microbial colonies growing therein, whereinthe image was obtained while illuminating only the back side of theculture device.

FIG. 6A is a detailed view of a portion of the image of FIG. 6.

FIG. 7 is a black and white image of the portion of the thin filmculture device of FIG. 6, wherein the image was obtained whileilluminating only the front side of the culture device.

FIG. 7A is a detailed view of a portion of the image of FIG. 7.

FIG. 8 is a graph of the relative intensities of red, green, and bluecomponents, respectively, of the pixels in the line scan of FIG. 6A.

FIG. 9 is a graph of the relative intensities of red, green, and bluecomponents, respectively, of the pixels in the line scan of FIG. 7A.

DETAILED DESCRIPTION

Detection and counting of microorganisms is a universal problem in manydiverse fields. Microorganisms occur in almost all foods, in water, inair, and on numerous surfaces and substances with which humans come incontact. Such microorganisms are often harmful and therefore must bemeasured and controlled.

A widely used practice for detecting the presence of microorganisms in asubstance (e.g., food, water, environmental residue) is to place asample of the substance to be tested, suitably prepared, in a culturedevice, and to allow the microorganisms to grow into colonies. Whencultured in such a medium, colonies become visible to the eye and can becounted. Each visible colony corresponds to one original microorganism.A method of the present disclosure is performed using such culturedevices for growing and counting microbial colonies. Typically, theculture device includes an aqueous nutrient medium and a matrix (e.g., agelling agent such as agar, guar gum, or pectin, for example) tomaintain separation of individual colonies. Many culture devices furtherinclude indicator compounds as discussed herein. Culture devices forgrowing and counting microbial colonies include, for example, agar Petridishes and thin film culture devices sold by 3M Company under thePETRIFILM trade name. PETRIFILM thin film culture devices are disclosedin numerous publications including, for example, U.S. Pat. Nos.5,364,766; 5,601,998; and 5,681,712; which are all incorporated hereinby reference in their entirety.

Many culture media, including typical agar culture media and culturemedia used in PETRIFILM plates, include indicator compounds to indicatethe presence of a microorganism. Indicator compounds include, forexample, pH indicators, chromogenic enzyme substrates, and redoxindicators. The indicator compounds, when converted directly orindirectly to a product, typically impart a color change to themicrobial colony and/or the culture medium surrounding the colony. Thecolor change often makes it easier to detect the presence of themicrobial colony in the culture medium (e.g., it improves the colorcontrast between the colony and the culture medium) and may the colorchange also may serve to differentiate a particular colony that reactswith a particular indicator compound from another microbial colony thatdoes not react with that indicator compound.

Many types of culture media for growing and differentiatingmicroorganisms include two or more indicator compounds. For example, theculture medium in a PETRIFILM E. coli Count Plate, when hydrated with anaqueous buffer and/or sample, contains a redox indicator(triphenyltetrazolium chloride, hereinafter “TTC”) and a chromogenicenzyme substrate (5-bromo-4-chloro-3-indolyl-β-D-glucuronide,hereinafter “X-gluc”). The TTC reacts with microbial cells to form areddish-colored formazan that stains the cell mass of any bacterialcolony that grows in the Gram-negative selective growth medium. Incontrast, the X-gluc reacts only with bacteria that, in addition tobeing able to grow in the selective growth medium, possessβ-D-glucuronidase enzyme activity (e.g., E. coli strains that possessβ-D-glucuronidase enzyme activity). Hydrolysis of X-gluc causes theformation of an indigo dye, which stains the cell mass of the colonyblue and forms a blue halo (i.e. zone of diffusion of the indicator)surrounding the colony having β-D-glucuronidase enzyme activity.

It is contemplated that the method of the present disclosure can be usedto distinguish microbial colonies on the basis of their reaction withone or more of a plurality of indicator compounds even if the microbialcells react with the indicator compounds to form products that aresubstantially the same color. This is possible when the product of oneof the indicator compounds remains associated with the cell mass of themicrobial colony and the product of another indicator compound diffusesinto the culture medium surrounding the cell mass of the colony.

In the method of the present disclosure, the sample is prepared,inoculated into the culture device, and incubated according toprocedures that are well known in the art. Sample preparation mayoptionally include dilution, enzymatic digestion, filtration, and/orsedimentation to reduce or remove nonmicrobial debris from the sampleprior to introducing the sample into (e.g., pour-plating) or onto (e.g.,surface-plating) the nutrient medium in the culture device.

After a sufficient incubation period at a temperature suitable for thegrowth of the microorganisms suspected of being present in the sample,microbial colonies can be detected and counted using an imaging systemto capture an image of microbial colonies in a culture device andapplying various image-analysis schemes. Examples of imaging systemsused to count and/or differentiate microbial colonies in a culturedevice can be found in International Publication No. WO 98/59314; andU.S. Pat. Nos. 7,298,885; 8,094,916; and 7,496,225; which areincorporated herein by reference in their entirety. Examples of imageanalysis schemes to detect and/or enumerate microbial colonies in aculture device can be found in U.S. Pat. Nos. 6,058,209 and 6,243,486,which are incorporated herein by reference in their entirety.

The present disclosure is directed to techniques for counting microbialcolonies in a culture device. The techniques can be used to improve theaccuracy of automated counts of microbial colonies in a culture device.The counting rules disclosed herein are typically stored ascomputer-executable software instructions, and are executed by aprocessor in a biological scanning system. Alternatively, the rules maybe implemented in hardware such as an application specific integratedcircuit (ASIC), a field programmable gate array (FPGA), or varioushardware components known in the art. The various rules described hereinmay be applied individually, or in any combination with other countingrules depending on the growth medium being scanned. In any case, byapplying the rules described herein, the accuracy of automated counts ofmicrobial colonies on a culture device can be improved.

In any embodiment, a method of the present disclosure employs a systemfor detecting and counting microbial colonies in a culture device.Systems for detecting and counting microbial colonies in a culturedevice are described, for example, in International Patent PublicationNos. WO 96/18720, WO 96/18167, WO 2005/062744, which are allincorporated herein by reference in their entirety.

FIG. 1 shows a perspective view of one embodiment of a system 20 fordetecting and counting microbial colonies in a culture device. Thesystem 20 comprises a scanner 21 coupled to an external computer 22which performs imaging analysis of the images generated by the scanner.External computer 22 may include, for example, a microprocessorprogrammed for image analysis of a culture device 24. External computer22 may comprise a personal computer (PC), desktop computer, laptopcomputer, handheld computer, workstation, tablet personal computingdevice, mobile device or the like. For example, software programs can beloaded on external computer 22 to facilitate image analysis of images ofculture device 24 generated by scanner 21.

Scanner 21 is coupled to external computer 22 via interface 25.Interface 25, for example, may comprise a Universal Serial Bus (USB)interface, a Universal Serial Bus 2 (USB2) interface, an IEEE 1394 FireWire interface, a Small Computer System Interface (SCSI) interface, anAdvance Technology Attachment (ATA) interface, a serial ATA interface, aPeripheral Component Interconnect (PCI) interface, a conventional serialor parallel interface, wireless connection or the like.

The culture device 24 optionally may include indicia 29, such as a barcode or other type of identification marking used to identify culturedevice 24. RFID tags, two-dimensional optically detectable codes, or thelike, may also be used as indicia. In any case, indicia 29 may identifythe type of microorganism being grown and tested on the culture device24. Scanner 21 can be designed to draw the culture device 24 intoscanner 21 to a first location and generate an image of indicia 29, andthen draw the culture device 24 to a second location and generate animage of the growth area 27. In this manner, images of indicia 29 andgrowth area 27 of the culture device can be generated by system 20.Alternatively, a single image may capture both indicia 29 and the growtharea 27. In either case, the scanning of indicia 29 can facilitateidentification of the type of plate being used so that one or moredesirable counting rules can be applied in an automated fashion.

By way of example, the culture device 24 may comprise a thin filmculture device sold by 3M under the trade name PETRIFILM plates. Culturedevice 24 can be utilized to facilitate the rapid growth and detectionof microorganisms commonly associated with food contamination,including, for example, aerobic bacteria, E. coli, coliform,enterobacteria, yeast, mold, Staphylococcus aureus, Listeria,Campylobacter, or the like. Culture devices generally comprise one typeof growth medium commonly used for biological growth and bacterialdetection and enumeration. The invention, however, may also be appliedwith a wide variety of other types of growth media.

In any embodiment, the thin film culture device can have a front sidethat comprises a transparent film cover sheet and a back side comprisesa translucent substrate, such as a PETRIFILM E. coli/Coliform CountPlate, a PETRIFILM Coliform Count Plate, and a PETRIFILMEnterobacteriaceae Count Plate, for example. Without being bound bytheory, it is believed the combination of a relatively thin (e.g.,approximately 1-2 mm thick) culture medium disposed between atranslucent film and a transparent film provides optical conditions thatare beneficial for distinguishing colonies according to the presentdisclosure.

In order to improve the accuracy of automated counts of microbialcolonies on a culture device, various aspects of the method of thepresent disclosure establishes rules that can be applied during imageprocessing. In other words, the rules described in greater detail belowcan form part of a colony counting algorithm executed in system 20. Therules may be used individually or in any combination with other imageanalysis rules (the counting rules described in International PatentPublication No. WO 2005/062744, which is incorporated herein byreference), depending on the type of growth medium being scanned and theproblems that may be encountered. Application of one or more of thecounting rules can improve a biological scanning system such as system20 by improving the accuracy of automated counts of microbial colonieson a growth medium such as a thin film culture device or the like.

FIG. 2 is a block diagram of a biological scanning system 30, which maycorrespond to system 20 (FIG. 1). System 30 includes an imaging device32 that generates one or more images of a growth medium and provides theimages to processor 34. Processor 34 is coupled to memory 36. Memory 36stores various processor-executable software instructions thatfacilitate image analysis of the images generated by imaging device 32.In particular, memory 36 stores one or more counting rules 37 which areapplied during image analysis to improve the accuracy of automatedcounts of microbial colonies on a culture device. Output device 38receives the results determined by processor 34 and provides the resultsto a user.

By way of example, imaging device 32 may comprise a 2-dimensionalmonochromatic camera for generating one or more images of a culturedevice. Various illuminators (not shown) may be used to illuminate thefront and back of culture device. For example, the illuminators canilluminate the culture device with one or more colors, and one or moreimages of the culture device can be generated by imaging device 32. Inaddition, a controller (not shown) can control a ratio of front-sideillumination to back side illumination for each image of the culturedevice. A non-limiting example of an imaging device that providesfront-side and back-side illumination that can be used to image a thinfilm culture device, optionally with a plurality of illumination colors,is described in U.S. Pat. No. 8,094,916, which is incorporated herein byreference in its entirety.

In an embodiment, a first image can be obtained using 100% of theillumination coming from illuminators illuminating the front side of theculture device and 0% of the illumination coming from illuminatorsilluminating the back side of the culture device and a second image canbe obtained using 0% of the illumination coming from illuminatorsilluminating the front side of the culture device and 100% of theillumination coming from illuminators illuminating the back side of theculture device. In another embodiment, for example, a first image can beobtained using 80% of the illumination coming from illuminatorsilluminating the front side of the culture device and 20% of theillumination coming from illuminators illuminating the back side of theculture device and a second image can be obtained using 20% of theillumination coming from illuminators illuminating the front side of theculture device and 80% of the illumination coming from illuminatorsilluminating the back side of the culture device. The ratio offront-side illumination to back-side illumination can be selected toprovide optimum contrast for a particular type of nutrient medium in theculture device.

In any embodiment of the method, the first image is produced whileilluminating the device with a first ratio (e.g., 100%:0%) of front-sideillumination to back-side illumination and the second image is producedwhile illuminating the device with a second ratio (e.g., 0%:100%) offront-side illumination to back-side illumination that is lower than thefirst ratio. In any embodiment, the first ratio can be greater than 1:1.In any embodiment, the second ratio can be less than 1:1.

It should be noted that “first image”, as used herein, refers to animage that is obtained while the culture device receives illuminationprimarily from the front side of the plate and “second image”, as usedherein refers to an image that that is obtained while the culture devicereceives illumination primarily from the back side of the plate. Animplied temporal order of obtaining the images is not intended by theuse of the terms “first image” and “second image”. Accordingly, a firstimage of a culture device can be obtained before or after a second imageof the culture device. In addition, one of the images (e.g., the firstimage or second image, respectively) does not need to be obtained by theimaging culture device immediately after obtaining the other image(e.g., the second image or first image, respectively). It is recommendedthe first and second images are obtained closely enough in time toobviate the possibility of significant biological changes (e.g., growthor enzyme activity) or physical changes (e.g., dehydration) occurringduring the intervening time between image acquisitions. Thus, in apreferred embodiment, the first image is obtained within about 30seconds of the time at which the second image is obtained.

A person having ordinary skill in the art will recognize that, in asystem wherein the imaging device is positioned facing the front side ofthe culture device and the illuminators are also positioned such thatthe illumination is directed at the front side of the culture device,the image produced by the imaging device substantially comprises lightthat is reflected from the culture device and the contents thereof. Inaddition, the person having ordinary skill in the art will alsorecognize that, in a system wherein the imaging device is positionedfacing the front side of the culture device and the illuminators arealso positioned such that the illumination is directed at the back sideof the culture device, the image produced by the imaging devicesubstantially comprises light that is transmitted by and/or refracted bythe culture device and the contents thereof.

The images are provided to processor 34 and may also be stored in memory36. In any case, the images are analyzed by applying counting rules 37in order to determine bacteria counts on the culture device. Theresolution of imaging device 32 may be approximately 155 pixels percentimeter. In that case, a one centimeter line in the image is 155pixels long.

Processor 34 may comprise a general-purpose microprocessor that executessoftware stored in memory 36. Alternatively, processor 34 may comprisean application specific integrated circuit (ASIC) or other specificallydesigned processor. In any case, processor 34 executes various countingrules 37 to improve the accuracy of automated counts of microbialcolonies on a culture device.

Memory 36 is one example, of a computer readable medium that storesprocessor executable software instructions applied by processor 34. Byway of example, memory 36 may comprise random access memory (RAM),read-only memory (ROM), non-volatile random access memory (NVRAM),electrically erasable programmable read-only memory (EEPROM), flashmemory, or the like. Counting rules 37 such as those described below,are stored in memory 36 and may form part of a larger software programused for image analysis.

Output device 38 typically comprises a display screen used tocommunicate results to a user. However, output device 38 could alsocomprise other types of devices such as a printer or the like. Outputdevice 38 may form part of a scanning unit, such as display (not shown),or may be external to the scanning unit, such as the display screen ofexternal computer 22 (FIG. 1).

FIG. 3 is a flow diagram illustrating a process of automated culturedevice analysis. As shown in FIG. 3, processor 34 receives one or moreimages of a culture device (step 41). Processor 34 invokes varioussoftware routines from memory 36 to count the microbial colonies on theculture device (step 42). For example, bacterial colonies may beidentified according to a characteristic color they produce afterreacting (i.e., directly or indirectly) with one or more indicatorcompounds in the nutrient medium. Other aspects of colony recognitionare discussed below. The software executed by processor 34 can allow foridentification of the growth area on the culture device and automatedcounting of bacterial colonies based on color changes in the growth areawhere the colonies have grown during incubation.

In accordance with the invention, processor 34 applies one or more rulesto improve the accuracy of the count of microbial colonies on the growthmedium (step 43). The rules may be applied individually or variouscombinations of rules may be used, depending on the type of culturedevice being analyzed. The rules may be individually invoked from memory36 or may form sub-routines of a larger image analysis software program.The rules may be applied individually or various sets of the rules maybe applied. If a set of rules are used, then the order in which therules are applied may be selected based on the type of plate beingscanned. The selected order for application of the rules may affect theend result. Various subsets of the rules may also be applied in anyorder, and the selected order for a subset of rules may also affect theend result.

FIG. 4 shows one embodiment of a method 100 according to the presentdisclosure. The method comprises the step 51 of obtaining a first imagewhile illuminating the front side of a culture device and the step 52 ofobtaining a second image while illuminating the back side of the culturedevice. The front side and back side of the culture device can beilluminated with an imaging system as disclosed herein. The method 100further comprises the step 53 of analyzing the first and second imagesto identify microorganism colonies in each image.

The first and second images are obtained so as to define objects in theimage in shades of at least one color. Thus, analyzing the first andsecond images to identify microorganism colonies in each image cancomprise identifying objects in the image as colonies according to imageanalysis methods that are well known in the art. For example, Weissdescribes techniques to identify microbial colonies in an image basedupon one or more criteria including object size, visibility, color,surface quality, and shape (U.S. Pat. No. 6,243,486, which isincorporated herein by reference in its entirety). As discussed above, amethod to detect a microbial colony that reacts with an indicatorcompound comprising TTC can be configured to detect a shade of the colorred and a method to detect a microbial colony that reacts with anindicator compound comprising 5-bromo-4-chloro-3-indolyl-β-D-glucuronidecan be configured to detect a shade of the color blue.

In a preferred embodiment of the method, a thin film culture devicehaving a second indicator that is a chromogenic enzyme substratecomprising an indolyl group is illuminated from the back-side and animage is taken with an illumination intensity and an integration time(i.e., exposure time) that results in near saturation of the image.Under the conditions of this preferred embodiment, the area in the imagedefined by the cell mass of a microbial colony can appear to besignificantly darker (i.e., permits less transmittance of light) thanthe zone of colored product (i.e., from the chromogenic enzymesubstrate) surrounding the colony. This facilitates the discriminationbetween the relatively smaller microbial colony from the relativelylarger colored zone of colored product (i.e., from the enzyme reaction)surrounding the colony. For example, under these conditions an imagingprocessor can readily discriminate the edges of the colony mass from theedges of colored product surrounding the colony based on the differencesin the color intensity of the regions (e.g., the difference inbrightness between the image of the colony and the image of a coloredzone surrounding the colony, if present).

Analyzing the first and second images to identify microorganism coloniesin each image further comprises identifying the location any coloniesdetected in the images. The locations will be used to identify andcompare measurable parameters associated with coincident colonies ineach image. The locations can be identified by X-Y coordinates in eachimage. Thus, in a preferred embodiment, both the first and second imagesare obtained without moving or otherwise handling the culture deviceafter the first image is obtained but before the second image isobtained. Alternatively, registration landmarks (e.g., two or morecorners of a PETRIFILM plate or registration marks made on any culturedevice) can be used to orient the images properly in order to determinecoincidental colonies in the first and second images.

After analyzing the first image and second image to identifycoincidental colonies, the first and second images are analyzed tocalculate a value related to a size parameter for each coincidentalcolony. The size parameter can be, for example, a mean colony diameter,a minimum colony diameter, a maximum colony diameter, or a colony area.Thus a first size parameter value is calculated for a particular colonyin the first image (FIG. 4, step 54) and a second size parameter valueis calculated for the corresponding coincident colony in the secondimage (FIG. 4, step 55).

The method 100 further comprises the step (56) of comparing the firstsize parameter value to the second size parameter value. If the firstsize parameter value calculated from a first image of a given colony iswithin a predetermined range (e.g., 80% to 120%, 90% to 110%, or 95% to105%) of the second size parameter value calculated from a second imageof the given colony, the colony is counted as a colony belonging to afirst group (e.g., group “A”: probable non-E. coli microorganisms). Inthis case, group “A” microorganisms react with a first indicatorcompound (e.g., TTC) that produces a nondiffusible product but does notreact with a second indicator compound (e.g.,5-bromo-4-chloro-3-indolyl-β-D-glucuronide) that produces a diffusibleproduct.

Conversely, if the first size parameter value calculated from a firstimage of a given colony is greater than the second size parameter value(e.g., at least 20% greater than, at least 50% greater than, or at least100% greater than the second size parameter value) calculated from thesecond image of the given colony, the colony is counted as a colonybelonging to a second group (e.g., group “B”: probable E. colimicroorganisms). In this case, group “B” microorganisms react with afirst indicator compound (e.g., TTC) that produces a nondiffusibleproduct and reacts with a second indicator compound (e.g.,5-bromo-4-chloro-3-indolyl-β-D-glucuronide) to produce a diffusibleproduct, which results in the first size parameter value being greaterthan the second size parameter value.

FIG. 5 is a flow diagram illustrating the rule for differentiatingcolonies into a plurality of colony types according to the presentdisclosure. As illustrated in FIG. 2, processor 34 invokes softwarestored in memory 36 to identify and map the location of colonies in thefirst image and second image (step 61). In particular, processor 34determines whether a colony identified in the first image maps to thesame location as a colony identified in the second image (step 62). Ifcoincidental colonies are found in the first and second images, theprocessor 34 calculates a size parameter value for the coincidentalcolonies and compares the values to determine whether they are different(step 63). If the values are not different (e.g., within a predeterminedrange, as discussed above), the colony in the culture device is countedas a first type (“Type A”, as indicated in step 64). If the values aredifferent (e.g., the value for the first image is less than the valuefor the second image), the colony in the culture device is counted as asecond type (“Type B”, as indicated in step 65). By way of example, aType “A” colony may react with TTC and not with5-bromo-4-chloro-3-indolyl-β-D-glucuronide in a PETRIFILM E.coli/Coliform Count Plate and, thus, would be identified as a non-E.coli colony. Conversely, a Type “B” colony may react with TTC and with5-bromo-4-chloro-3-indolyl-β-D-glucuronide in a PETRIFILM E.coli/Coliform Count Plate and, thus, would be identified as an E. colicolony.

Analyzing the images can include RGB (red/green/blue) image processingalgorithms. Alternatively, or additionally, analyzing the images caninclude HSI (hue, saturation, and intensity), HSL (hue, saturation, andlightness), HSV (hue, saturation, and value) algorithms, or combinationsthereof.

FIG. 6 shows a black and white image of a portion of a thin film culturedevice having two types of microbial colonies growing therein. The imagewas obtained while illuminating only the back side of the culturedevice, as described in Example 1. Within the growth area is a pluralityof microbial colonies, including colonies of two different types “A” and“B”, respectively. Colonies belonging to type “A” react with a firstindicator compound that reacts with and stains the cell mass of thebacterial colony. Colonies belonging to type “B” interact with the firstcompound and, in addition, also react with a second indicator compoundthat imparts a color change to the microbial colony and/or the culturemedium surrounding the colony. The diameters of representative coloniesbelonging to type A (first colony 74) and type B (second colony 75) weredetermined according to the technique described in Example 1. The data(listed in Table 1 below) show the diameter of first colony 74 in theback-illuminated image is 22 pixels and the diameter of second colony 75in the back-illuminated image is 15 pixels. Thus, FIG. 6 shows that,with this type of illumination (i.e., back-side illumination), a sizeparameter (i.e., the colony diameter) of representative coloniesbelonging to both types (A and B) may have approximately the samemagnitude. Also shown in FIG. 6A is a path 91 of a line scan of aportion of the growth area that includes at least one microbial colony.

FIG. 7 shows a black and white image of the same portion of the thinfilm culture device shown in FIG. 6. The image was obtained whileilluminating only the front side of the culture device, as described inExample 1. The diameters of a first colony 74 and second colony 75 weredetermined according to the technique described in Example 1. The datashow the diameter of first colony 74 in the front-illuminated image is22 pixels and the diameter of second colony 75 in the front-illuminatedimage is 45 pixels. Thus, FIG. 7 shows that, with this type ofillumination (i.e., front-side illumination), a size parameter (i.e.,the colony diameter) of representative colonies belonging to both types(A and B) may have different magnitudes (e.g., substantially differentmagnitudes). Also shown in FIG. 7A is a path 92 of a line scan of aportion of the growth area that includes at least one microbial colony.Path 92 corresponds to the same pixels as those in path 91 of FIG. 6A.

In order to identify the presence and location of colonies in a back-litimage and to identify the presence and location of microbial colonies ina front-lit image, image-analysis algorithms often analyze the pixels inthe digital image line-by-line, comparing the color hue and/or colorintensity of a first pixel or first group of pixels to the color hueand/or color intensity of a second pixel proximate the first pixel orsecond group of pixels proximate the first group of pixels. This type ofcomparison permits the algorithm to recognize color and/or intensityshifts that may indicate the edge of a microbial colony or other objectin the image. FIG. 8 shows a graph of the transmitted pixel intensitiesfor red, green, and blue obtained from pixels along the path 91 in theback-lit image of FIG. 6A. Each of the two negative peaks (“1”, and “2”,respectively) show that the centers of the respective colonies aredarker than the proximate background of the growth medium, therebyclearly indicating the presence of two distinct colonies. FIG. 9 shows agraph of the reflected color intensities for red, green, and blueobtained from pixels along the path 92 in the front-lit image of FIG.7A. FIG. 9 shows smaller differences in pixel intensities between thepixels comprising the colony centers and the pixels comprising thegrowth medium adjacent the colonies.

The use of a scanning system with a counting rule for differentiatingmicrobial colonies has been described. The counting rule can be used ina scanning system to improve the accuracy of automated counts ofmicrobial colonies on a culture device.

The techniques have been described as being software-implemented. Inthat case, a computer readable medium stores processor executableinstructions that embody one or more of the rules described above. Forexample, the computer readable medium may comprise non-transitorycomputer readable media such as random access memory (RAM), read-onlymemory (ROM), non-volatile random access memory (NVRAM), electricallyerasable programmable read-only memory (EEPROM), flash memory, or thelike. The computer readable medium may also comprise a non-volatilememory such as a CD-ROM used to deliver the software to customers. Also,the computer readable medium may comprise an electromagnetic carrierwave, e.g., for delivering the software over a network such as theinternet.

The same techniques, however, may also be implemented in hardware.Example hardware implementations include implementations within anapplication specific integrated circuit (ASIC), a field programmablegate array (FPGA), specifically designed hardware components, or anycombination thereof. In addition, one or more of the techniquesdescribed herein may be partially executed in hardware, software orfirmware.

Embodiments

Embodiment A is a method, comprising:

using an imaging device to produce a first image of a thin film culturedevice, the culture device having a front side having a transparent filmcover sheet and a back side having a translucent substrate;

-   -   wherein the first image is produced while providing illumination        to the front side of the device;    -   wherein the culture device comprises first and second indicator        compounds;        -   wherein the first indicator compound is converted by a            microorganism to a first product having a first color;        -   wherein the second indicator compound is converted by a            microorganism to a water-diffusible second product that            forms a second color;

using the imaging device to produce a second image of the thin filmculture device, wherein the second image is produced while providingillumination to the back side of the device;

analyzing the first and second images to identify microorganism coloniesin each image;

analyzing the first image to calculate a first value of a size parameterfor a colony at a particular location in the culture device;

analyzing the second image to calculate a second value of the sizeparameter at the particular location in the culture device; and

comparing the first value to the second value.

Embodiment B is the method of Embodiment A, wherein the first image isproduced while illuminating the device with a first ratio of front-sideillumination to back-side illumination, wherein the second image isproduced while illuminating the device with a second ratio of front-sideillumination to back-side illumination that is lower than the firstratio.

Embodiment C is the method of Embodiment B, wherein the first ratio isgreater than 1:1.

Embodiment D is the method of Embodiment B, wherein the first ratio isabout 100%:0%.

Embodiment E is the method of any one of Embodiments B through D,wherein the second ratio is about 0%:100%.

Embodiment F is the method of any one of the preceding Embodiments,further comprising using the first or second image to count a number ofmicroorganism colonies in the culture device.

Embodiment G is the method of any one of the preceding Embodiments,further comprising using the first and second image to count a number offirst type colonies and count a number of second type colonies in theculture device.

Embodiment H is the method of Embodiment G, wherein the first typecolonies convert the first indicator to the first product.

Embodiment I is the method of Embodiment G or Embodiment H, wherein thesecond type colonies convert the second indicator to the second product.

Embodiment J is the method of any one of the preceding Embodiments,wherein the first indicator compound comprises a tetrazolium dye.

Embodiment K is the method of any one of the preceding Embodiments,wherein the second indicator compound comprises a chromogenic enzymesubstrate that includes an indolyl group.

Embodiment L is the method of any one of the preceding Embodiments,wherein the size parameter is an observed colony diameter.

Embodiment M is the method of Embodiment L, wherein the colony diameteris a colony minimum diameter.

Embodiment N is a computer readable medium comprising computer readableinstructions that, when executed by a processor, cause a culture platescanning system comprising the processor to:

obtain a first image of a thin film culture device, wherein the firstimage is produced while illuminating the device with a first ratio offront-side illumination to back-side illumination;

obtain a second image of the thin film culture device, wherein thesecond image is produced while illuminating the device with a secondratio of front-side illumination to back-side illumination that is lowerthan the first ratio;

analyze the first and second images to identify microorganism coloniesin each image;

analyze the first image to calculate a first value of a size parameterfor a colony at a particular location in the culture device;

analyze the second image to calculate a second value of the sizeparameter at the particular location in the culture device; and

compare the first value to the second value.

Embodiment O is the computer readable medium of Embodiment N furthercomprising computer readable instructions that, when executed in theprocessor, cause the system to use the first or second image to count anumber of microorganism colonies in the culture device.

Embodiment P is the computer readable medium of Embodiment N furthercomprising computer readable instructions that, when executed in theprocessor, cause the system to use the first and second image to count anumber of first type colonies and count a number of second type coloniesin the culture device.

Embodiment Q is a method, comprising:

using an imaging device to produce a first image of a culture device,the culture device having a front side and a back side opposite thefront side;

-   -   wherein the first image is produced while providing illumination        to the front side of the device;    -   wherein the culture device comprises first and second indicator        compounds;        -   wherein the first indicator compound is converted by a            microorganism to a first product having a first color;        -   wherein the second indicator compound is converted by a            microorganism to a water-diffusible second product that            forms a second color;

using the imaging device to produce a second image of the thin filmculture device, wherein the second image is produced while providingillumination to the back side of the device;

analyzing the first and second images to identify microorganism coloniesin each image;

analyzing the first image to calculate a first value of a size parameterfor a colony at a particular location in the culture device;

analyzing the second image to calculate a second value of the sizeparameter at the particular location in the culture device; and

comparing the first value to the second value.

Embodiment R is the method of Embodiment Q, wherein the first image isproduced while illuminating the device with a first ratio of front-sideillumination to back-side illumination, wherein the second image isproduced while illuminating the device with a second ratio of front-sideillumination to back-side illumination that is lower than the firstratio.

Embodiment S is the method of Embodiment R, wherein the first ratio isabout 100%:0%.

Embodiment T is the method of Embodiment R or Embodiment S, wherein thesecond ratio is about 0%:100%.

Embodiment U is the method of any one of Embodiments Q through Twherein, while the first and second images are produced, an opticaldiffuser is disposed proximate the back side of the culture device.

Embodiment V is the method of any one of Embodiments Q through U,further comprising using the first or second image to count a number ofmicroorganism colonies in the culture device.

Embodiment W is the method of any one of Embodiments Q through V,further comprising using the first and second image to count a number offirst type colonies and count a number of second type colonies in theculture device.

Embodiment X is the method of Embodiment W, wherein the first typecolonies convert the first indicator to the first product.

Embodiment Y is the method of Embodiment W or Embodiment X, wherein thesecond type colonies convert the second indicator to the second product.

Embodiment Z is the method of any one of claims Q through Y, wherein thefirst indicator compound comprises a tetrazolium dye.

Embodiment AA is the method of any one of Embodiments Q through Z,wherein the second indicator compound comprises a chromogenic enzymesubstrate that includes an indolyl group.

Embodiment BB is the method of any one of Embodiments Q through AA,wherein the first ratio is about 100%:0%, wherein the second ratio isabout 0%:100%.

Embodiment CC is the method of any one of Embodiments Q through BB,wherein the size parameter is a colony diameter.

Embodiment DD is the method of Embodiment CC, wherein the colonydiameter is a colony minimum diameter.

EXAMPLES Method for Detecting E. coli Colonies

Tryptic Soy Broth (TSB, Catalog # K89) was obtained from HardyDiagnostics (Santa Maria, Calif.). Microbial strains E. coli (ATCC25922), E. coli (3M-FR4), Salmonella enterica (ATCC 51812) andEnterobacter amnigenus (ATCC 51898) were obtained from MicrobiologicsInc (St Cloud, Minn.). An overnight TSB culture was prepared for eachmicrobial strain. Thin film culture devices (3M PETRIFILM E.Coli/Coliform Count (EC) Plates) and Butterfield's Phosphate Buffer wereboth obtained from the 3M Company (St. Paul, Minn.).

Dilutions from overnight cultures of each strain were prepared inButterfield's Phosphate Buffer to yield approximately 25 colony-formingunits (CFU) per mL. The 3M PETRIFILM plates were inoculated by liftingthe transparent film cover sheet, pipetting 1 mL of the diluted samplein the center of the coated bottom film, and replacing the cover sheet.The sample was uniformly spread to the desired surface area(approximately 20 cm²) using the spreading device provided by themanufacturer (3M). Inoculated plates were incubated at 35° C. for 24hours.

The colonies on the PETRIFILM culture plate were imaged and identifiedusing a culture device imaging system. The imaging system contained acentrally positioned glass platen (White Flashed Opal Glass) that servedas a platform for placement of the culture plate. The culture plate wasilluminated on the front side using two separate sets of light emittingdiodes (each set containing two red LEDs, two green LEDs, and two blueLEDs) One set was positioned above to the left (relative to thelongitudinal dimension) of the culture plate and the other set waspositioned above and to the right (relative to the longitudinaldimension of the culture plate). Light from the LEDs positioned abovethe culture device was directed away from the culture device and into alight-diffusing reflective surface, which directed a substantiallyuniform illumination pattern onto the front side of the culture plate.Similarly, the culture plate was illuminated on the back side using twoseparate sets of light emitting diodes (each set containing two redLEDs, two green LEDs, and two blue LEDs). One set was positioned belowand to the left (relative to the longitudinal dimension) of the cultureplate and the other set was positioned below and to the right (relativeto the longitudinal dimension of the culture plate). Light from the LEDspositioned below the culture device was directed away from the culturedevice and into a light-diffusing reflective surface, which directed asubstantially uniform illumination pattern onto the back side of theglass platen (described above), and created a uniform illuminationpattern on the back side of the culture plate.

An Aptina Model MT9P031 CMOS imaging sensor (Aptina Imaging, San Jose,Calif.) was orthogonally-positioned above the platform in order to takeimages of the culture plate. The imaging sensor and platform wereadjusted so that the culture plate was positioned within the focal planeof the sensor. The culture plate was oriented on the platform so thatfront side (transparent film side) of the culture plate faced theimaging sensor. A black cover was used to isolate the imaging devicefrom room light. The image exposures were selected so that, in theacquired images, less than about 10% of the pixels in a histogram of allof the image pixels were saturated. A first image was taken using onlyillumination from the front side of the culture plate and a second imagewas taken using only illumination from the back side of the cultureplate. Both images were taken with the culture plate being maintained inexactly the same position on the platform (i.e., the plates were notmoved from the platform until both images were acquired). This allowedfor the identification of coincidental colonies in the two images bymatching the corresponding X-Y coordinate positions.

The two images were analyzed for colony type using ImagePro Plussoftware (Media Cybernetics, Rockville, Md.). For each image, the sizeof an individual colony was determined by measuring the colony diameter.The imaging program analyzed for changes in red, green, and blue pixelintensities observed along a line of pixels incorporating the longestdimension of the suspect colony's image. The pixel positions thatdefined a change in intensity relative to the local background were usedto mark the margins of the colony image and to measure the colonydiameter (diameter distance was reported as the number of pixels locatedbetween the pixel points marking the colony margins). The diametermeasurements for coincidental colonies in the two images were thencompared to determine the colony type. The results for threerepresentative colonies on the culture plate are presented in Table 1.Colonies designated 1 and 2; respectively correspond to colony 74 andcolony 75, respectively in FIGS. 6 and 7.

TABLE 1 Colony Colony Colony Diameter Diameter Diameter (Front-side(Back-side Measured Colony Illumination) Illumination) Difference ColonyDesignation (pixels) (pixels) (pixels) Type** 1 22 22 0 non-E. coli 2 4515 30 E. coli 3 100* 22 78 E. coli 4 100* 23 77 E. coli *Colony 3 andColony 4 were both surrounded by a single, diffuse blue halo (i.e., adiffusion zone diffusion of the product of the chromogenic indicatorsurrounding the colony). Thus, colonies 3 and 4 appeared as a singlelarge colony using front-side illuminated image. However, the back-sideilluminated image revealed that there were actually two separatecolonies whose blue zones had merged together in the plate **Colonytypes were determined based upon the difference between the observedcolony diameters measured using front-side-illuminated images comparedto the colony diameters measured using back-side-illuminated images.

In any case, various modifications may be made without departing fromthe spirit and scope of the invention. For example, one or more of therules described herein may be used with or without other rules andvarious subsets of the rules may be applied in any order, depending onthe desired implementation. These and other embodiments are within thescope of the following claims.

The invention claimed is:
 1. A non-transitory computer readable medium comprising computer readable instructions that, when executed by a processor, cause a culture plate scanning system comprising the processor to: obtain a first image of a thin film culture device, wherein the first image is produced while illuminating the device with a first ratio of front-side illumination to back-side illumination; obtain a second image of the thin film culture device, wherein the second image is produced while illuminating the device with a second ratio of front-side illumination to back-side illumination that is lower than the first ratio; analyze the first and second images to identify microorganism colonies in each image; analyze the first image to calculate a first value of a size parameter for a colony at a particular location in the culture device; analyze the second image to calculate a second value of the size parameter at the particular location in the culture device; and compare the first value to the second value.
 2. The non-transitory computer readable medium of claim 1 further comprising computer readable instructions that, when executed in the processor cause the system to use the first or second image to count a number of microorganism colonies in the culture device.
 3. The non-transitory computer readable medium of claim 1 further comprising computer readable instructions that, when executed in the processor, cause the system to classify the colony at the particular location as a colony belonging to a microorganism group.
 4. The non-transitory computer readable medium of claim 3 further comprising computer readable instructions that, when executed in the processor, cause the system to count a number of colonies belonging to the microorganism group.
 5. The non-transitory computer readable medium of claim 4 further comprising computer readable instructions that, when executed in the processor, cause the system to use the first and second image to count a number of colonies that belong to a first group and count a number of colonies that belong to a second group. 